1. Field of the Invention
The present invention relates to a three-dimensional structure of crystallization of the complex of NanR which is a key pathogenic regulatory protein of Vibrio vulnificus and ManNA6P which is a NanR regulator. Further, the present invention relates to a modified NanR protein, a polynucleotide encoding the protein, a vector including the polynucleotide, and a transformant including the vector. Furthermore, the present invention relates to a method for screening a substance regulating interaction between NanR and the transcriptional control region of nan operon which is a gene cluster regulated by NanR, or a substance regulating interaction between NanR and ManNAc-6P, and an antibacterial composition including the screened substance.
2. Description of the Related Art
When the human body is infected with pathogenic bacteria, the pathogenic bacteria encounter competition with the gut intestinal flora for nutrients in the host (human body). Therefore, these pathogenic bacteria overcome the nutritionally adverse environment by utilizing alternative carbon sources in the gut, and endeavor to survive. The intestinal tract of the human body is protected by a mucus layer containing a glycosylated protein mucin that is composed of 85% carbohydrate. Pathogenic bacteria such as Vibrio cholera and Vibrio vulnificus (V. vulnificus) have most likely evolved elaborate systems for catabolic utilization of N-acetylneuraminic acid (Neu5Ac), which is the most abundant sialic acids constituting mucin, as an alternative energy source.
When the human body is infected with these bacteria, these bacteria overexpress nan genes encoding enzymes essential for Neu5Ac catabolism and membrane transport proteins required for intracellular transport of Neu5Ac in the intestine so as to utilize Neu5Ac as an energy source, and thus they exert their pathogenicity through survival and growth, indicating that Neu5Ac catabolism of pathogenic microorganisms is directly correlated with their pathogenicity.
V. vulnificus is a pathogenic bacterium which usually enters the body through traumatic injury or ingestion of undercooked or contaminated sea food, and a life-threatening foodborne enteropathogen which causes septicemia in patients with liver disease or diabetes or in immunocompromised individuals, and the septicemia is associated with a mortality greater than 50% within 48 hours. Before entering the bloodstream, V. vulnificus survives and colonizes the small intestine. The present inventors recently demonstrated that NanR protein of V. vulnificus is a transcriptional repressor of the nan operon which is a cluster of genes encoding the transporter for intracellular absorption of Neu5Ac and essential catabolic enzymes for Neu5Ac, respectively. They also showed that N-acetylmannosamine 6-phosphate (ManNAc-6P), the catabolic intermediate of Neu5Ac, selectively binds to NanR and induces expression of the nan genes. However, the molecular mechanisms underlying regulation of the nan genes by interaction between NanR protein and ManNAc-6P has not been clarified yet.
On the other hand, recent studies change their approach to development of therapeutic agents from a random approach to exploration of a number of therapeutic candidates to a new approach to development of therapeutic agents with improved target specificity and efficacy by exploration of key target proteins, investigation of three-dimensional structure and function of the target proteins, and designing and development of drug candidates through specific and efficient protein engineering. Therefore, to design and develop selective and specific drug candidates, it is essential that production and crystallization of a large amount of a highly pure, stable protein are conducted and then its three-dimensional structure is investigated. Three-dimensional structure of the NanR protein, in particular, a complex of the NanR protein and ManNAc-6P, for all its importance, has not been revealed yet, because it is difficult to crystallize in stable form.
Accordingly, the present inventors have made many efforts to investigate three-dimensional structure of the NanR protein. As a result, they prepared a crystal of NanR protein and its ligand ManNAc-6P complex, and investigated interaction between the NanR protein and its regulatory ligand at the atomic level with high resolution, thereby completing the present invention.